Milk Science Lecture 5: Casein Properties PDF

Primary structure of casein – All caseins (polar and non-polar residues) are not uniformly distributed but occur in clusters, giving hydrophobic region and hydrophilic regions. – This feature makes the caseins good emulsifiers. (Emulsion is a mixture of two or more liqui...

Primary structure of casein – All caseins (polar and non-polar residues) are not uniformly distributed but occur in clusters, giving hydrophobic region and hydrophilic regions. – This feature makes the caseins good emulsifiers. (Emulsion is a mixture of two or more liquids that are normally non-mixable)  Casein carbohydrate – αs1-, αs2, β-caseins contain no carbohydrate – κ-casein contains about 5% carbohydrate (N-acetylneuraminic acid (sialic acid), galactose and N-acetylgalactosamine), Glycosylation – The carbohydrate provide on κ-casein quite high solubility and hydrophilicity  Casein phosphorus – Milk contains about 900 mg phosphorus/L – Casein contains about 0.85% phosphorus inorganic: soluble and colloidal phosphates organic: phospholipids, casein and sugar phosphates, nucleotides – Phosphorus is important: 1) nutritionally because it can bind large amounts of Ca2+, Zn2+ and other metals 2) increases the solubility of caseins 3) high heat stability of casein 4) important in the coagulation of rennet-altered casein – The phosphorus is covalently bound to the protein – Phosphate is bound mainly to serine – Phosphorylation occurs in the Golgi membrane  Secondary and tertiary structures of casein Caseins have relatively little secondary or tertiary structure, probably due to the presence of high levels of proline residues, especially in β-casein, which disrupt α-helix and β-sheet. The lack of secondary and tertiary structures is probably significant for the following reasons: – 1) The caseins are readily susceptible to proteolysis This has obvious advantages for the digestibility of the caseins, nutritionally. The caseins are also readily hydrolysed in cheese, which is important for the development of cheese flavor and texture – 2) The caseins adsorb readily at air-water and oil-water interfaces due to a) their open structure b) relatively high content of non-polar amino acid residues c) the uneven distribution of amino acids. This gives the caseins very good emulsifying and foaming properties, which are widely exploited in the food industry. – 3) The lack of higher structures probably explains the high stability of the caseins to denaturing agents, including heat  Molecular size of casein – All the caseins are relatively small molecules, ranging in molecular weight from about 20 to 25 kDa Hydrophobicity – The caseins are often considered to be rather hydrophobic molecules Influence of Ca2+ on caseins – αs1- and αs2-caseins are insoluble in calcium-containing solutions – β-casein is soluble at high concentrations of Ca2+ (0.4M) at temperatures below 18°C – κ-casein is soluble in Ca2+ at all concentrations.  Action of rennets on casein – κ-casein is the only major casein hydrolysed by rennets during the primary phase of milk coagulation (in cheese making) Casein association – κ-casein is present largely as disulphide-linked polymers – At 4 °C, β-casein exists in solution as monomers; at 8.5°C, 20 monomers chains – αs1-casein polymerizes to form tetramers of molecular mass, 113 kDa – The major caseins interact with each other and, in the presence of Ca2+ these associations lead to the formation of casein micelles. Casein Micelle Structure - Composition and general features – About 95% of the casein exists in milk as large colloidal particles, known as micelles (colloid is a substance dispersed throughout another substance) – On a dry matter basis, casein micelles contain 94% protein and 6% low molecular weight species (calcium, magnesium, phosphate and citrate) – The micelles are highly hydrated, binding about 2.0g of H2O/g protein – Casein micelles are generally spherical in shape, with diameters ranging from 50 to 500nm (average 120nm) – There are 1014-1016 micelles/ml milk; they are roughly two micelle diameters (240nm) apart. – The surface area of the micelles is very large; the surface properties of the micelles are critical to their behavior. – The white color of milk is due largely to light scattering by the casein micelles 2) Stability of micelles – stable to the normal processes (very stable at high temperatures) – can be sedimented by ultracentrifugation and re-dispersed readily by mild agitation. – stable to commercial homogenization – stable to high [Ca2+], up to at least 200mM at temperatures up to 50°C – aggregate and precipitate from solution when the pH is adjusted to the isoelectric point of caseins (pH4.6) – Many proteinases catalyse the hydrolysis of a specific bond in κ-casein – destabilized by 40% ethanol at pH 6.7 and by lower concentrations if the pH is reduced – destabilized by freezing due to a decrease in pH and an increase in the [Ca2+] in the unfrozen phase of milk 3) Principal micelle characteristics – Knowledge of micelle structure is important because the stability and behavior of the micelles are central to many dairy processing operations, (e.g., cheese manufacture, stability of sterilized sweetened-condensed and reconstituted milks and frozen products) – κ-Casein, which represents about 15% of total casein, is a critical feature of micelle structure and stability. Stabilize calcium-sensitive αs1, αs2 and β-caseins (85% of total casein) – Micelle has a porous structure in which the protein occupies about 25% of the total volume – Chymosin and similar proteinases specifically hydrolyse most of the micellar κ- casein – When heated in the presence of whey proteins (as in normal milk), κ-casein and β- lactoglobulin interact to form a disulphide-linked complex → modify rennet coagulability and heat stability – The micelles can be destabilized by alcohols and acetone-electrostatic interactions in micelle structure – As the temperature is lowered, β-casein dissociate from the micelles 4) Micelle structure- subunit (submicelles) Schmidt, Walstra, Ono: The κ-casein content of the submicelles varies and that the κ-casein deficient submicelles are located in the interior of the micelles with the κ-casein-rich submicelles concentrated at the surface Some αs1, αs2 and β-caseins are also exposed on the surface  Because the micelles are closely packed, inter-micellar collisions are frequent; however, the micelles do not normally remain together after collisions The micelles are stabilized by two principal factors: – (1) a surface (zeta) potential of -20mV at pH 6.7, which is probably too small for colloidal stability (0에 가 까울수록 안정성 떨어짐) – (2) steric stabilization (입체적 안전성) due to the protruding κ-casein hairs Zeta potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle Liquid system에서 분산되어 있는 파티클 (콜로이드 상태)은 전기적인 charge를 가짐 수용액에서 이온을 가져와서 생김 전기적으로 음성 또는 양성을 띰  On a commercial scale, whey protein-rich products are prepared by: 1. Ultrafiltration after acid or rennet whey to remove varying amounts of lactose, and spray-drying to produce whey protein concentrates (30-80% protein) 2. Ion-exchange chromatography: proteins are adsorbed on an ion exchanger, washed free of lactose and salts and then eluted by pH adjustment. The elute is free of salts by ultrafiltration and spray-dried to yield whey protein isolate, containing about 95% protein 3. Demineralization by electrodialysis and/or ion exchange, thermal evaporation of water and crystallization of lactose 4. Thermal denaturation, recovery of precipitated protein by filtration/centrifugation and spray-drying, to yield lactalbumin which has very low solubility and limited functionality